The present invention is a method and composition using chondroitinase B and chondroitinase AC, glycosaminoglycan degrading enzymes, to inhibit the formation of fibrotic tissue.
Proteoglycans on the cell surface and in the extracellular matrix contain variable glycosaminoglycan chains, which include heparan sulfate and chondroitin sulfates A, B, or C. While some proteoglycans contain only one type of glycosaminoglycan, others contain a mixture of heparan and chondroitin sulfates (Jackson et. al., Physiol. Rev. 71:481–530,1991). Extracellular proteoglycans form a structural framework for cells and tissues, and together with cell-associated proteoglycans, have major functions in regulating cell adhesion, migration, and proliferation. The functions of proteoglycans and their component parts have been extensively studied, with much of the emphasis on the roles of heparin and heparan sulfate on cell metabolism (Kjellen, L., and Lindahl, U. (1991) Ann. Rev. Biochem. 60:443–475; Vlodavsky, et al. (1995) Thrombosis Haemostasis 74:534–540; Yayon, et al. (1991) Cell 64:841–848)). Much less is known about the biological activities of proteoglycans containing chondroitin sulfate glycosaminoglycans, and in particular, their effects on cell proliferation.
Two inhibitors of glycosaminoglycan synthesis, chlorate and beta-xyloside, have been used to examine the relative contributions of heparan and chondroitin sulfate proteoglycans to control of the cell cycle (Keller, et al. (1989) Biochem. 28:8100–8107; Miao, et al. (1995) J. Cell. Biochem. 57:713–184; Schwartz, N. B. (1977) J. Biol. Chem. 252:6316–6321). However both of these compounds inhibit the expression of all types of sulfated glycosaminoglycans. There are currently no inhibitors which can selectively block the synthesis or expression of chondroitin sulfate A, B or C. However, specific glycosaminoglycan lyases which can remove either heparan or chondroitin sulfates A, B or C from cells are available. Chondroitinases have been isolated from several bacterial species: Flavobacterium heparinum, Aeromonas sp., Proteus vulgaris, Aurebacterium sp. and Bacillus thetaiotamicron (Linhardt, et al. (1986) Appl. Biochem. Biotech. 12:135–175; Linn, et al. (1983) J. Bacteriol. 156:859–866; Michellacci, et al. (1987) Biochim. Biophys. Acta. 923:291–301; and Sato, et al. (1986) Agric. Biol. Chem. 50:1057–1059).
Most studies examining the activities of chondroitin sulfate proteoglycans (Lyon, et al. (1998) J. Biol. Chem. 273:271–278; Maeda, et al. (1996) J. Biol. Chem. 271:21446–21452; Milev, et al. (1998) J. Biol. Chem. 273:21439–21442; Rapraeger, 1989, and Schmidt, et al. (1992) J. Biol. Chem. 267:19242–19247) have utilized one such enzyme, chondroitinase ABC (from Proteus vulgaris, Yamagata, et al. (1968) J. Biol. Chem. 243:1523–1535) which degrades all chondroitin sulfates (chondroitin sulfate A, chondroitin sulfate C and chondroitin sulfate B). Since chondroitinase ABC acts on more than one type of chondroitin sulfate, it is not possible to determine the biological activity of the individual types of chondroitin sulfates using this enzyme.
Evidence for a role of chondroitin sulfate A or B or C proteoglycans in cell proliferation includes data which shows upregulation during rapid cell proliferation, as occurs in wound healing (Penc and Gallo (1998) J. Biol. Chem. 273:28116–28121; Yeo, et al. (1991) Amer. J. Pathol. 138:1437–1450) and down regulation in quiescent cells (Tao et. al. (1997) Atherosclerosis 135:171–179). Such studies suggest that there may be a relationship between the secretion and expression of cell surface chondroitin sulfate proteoglycans and cell proliferation.
Recent studies in wound healing have found that chondroitin sulfate B proteoglycans are present in high concentration in the fluid of healing wounds, and that addition of these proteoglycans to a wounded area may promote healing (Penc and Gallo, 1998). Although the mechanism of action of the chondroitin sulfate B in wound healing is unknown, it is possible that these proteoglycans may directly or indirectly affect cell proliferation.
It is therefore an object of the present invention to provide a method and compositions for treatment of conditions associated with abnormal formation of fibrous tissue, through modification of proteoglycans.
It is another object of the present invention to provide a method and compositions to modulate collagen synthesis, decrease TGFbeta production, decrease fibroblast proliferation or migration, release chondroitin sulfate proteoglycans from cells, and decrease growth factor binding sites on fibroblasts.
It is a further object of the present invention to provide a method and compositions to treat disorders which involve hyperproliferation of fibroblasts such as scleroderma, psoriasis, keloids, pulmonary fibrosis and surgical adhesions.